GSM7977849: SK-LMS-1 cells, dsRNA RIP senescent RepA; Homo sapiens; RIP-Seq
Total RNA was extracted with Tri Reagent (Molecular Research Center) from a pellet of 35*106 SK-LMS-1HDAC4-/-/HDAC4PAM-ER cells grown for 96h with or without 4-OHT. Three biological replicates were pulled and subjected to two independent IP. IP was performed O/N in Polysomal Lysis Buffer in the presence of 3 μg of J2 antibody (Scicons). Immunocomplexes were purified by incubating O/N each sample with 16 μl magnetic protein A/G covered Dynabeads (ThermoFisher). After 3 washes of the collected immunocomplexes, RNA was recovered by phenolchloroform extraction/ethanol precipitation and resuspended in 10 μl RNase-free water (Ambion). RNA concentration, integrity and purity were tested by Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). 10ng RNA were DNAseI digested (NEB), rRNA depleted (using Ribo-Zero method) and reverse transcribed obtaining cDNA. Adapter were ligated to fragmented cDNA after end-repair and A-Tailing following DNBSEQ protocol (MGI-Tech). 18 rounds of PCR amplification were performed to enrich the cDNA fragments. Then the PCR products were purified with Ampure XP Beads (Agencourt). ssCirDNA was obtained after denaturation and circularization. DNA balls were generated from each library following BGI specifications (SOP-SS-025)